Material and methods
We used inbred lines described in Łukasiewicz et al. (2020), that were
inbred via 4 generations of brother-sister mating and continued
inbreeding for 6 more generations. In brief, mites collected from onion
bulbs in Mosina, Wielkopolska, Poznań Poland, and inbred lines were
initiated from the first generation of which hatched in the lab. The
proportion of both male phenotypes used for crosses reflected that in
the original population, giving rise 26 inbred lines founded by fighter
males (fighter lines henceforth), and 15 inbred lines founded by
scrambler males (scrambler lines). The consecutive generations were
established by mating a randomly selected female with her sibling male
of the same morph as the male founder. Choosing the same morph as the
founder to establish each generation allowed us to maintain differences
in morph proportions between F and S inbred lines throughout the
inbreeding experiment (Fig. S1).
We established outbred populations enriched for scrambler or fighter
genes (F and S populations henceforth) by mixing 12 inbred lines with
the highest ratio of the desired morph after 10 generations of
brother-sister mating described above. In this way we ensured that genes
for the desired morph were also overrepresented among females.
Furthermore, using inbred lines ensured effective population sizes of F
and S populations were similar (i.e. contained about 24 genes randomly
sampled from the original wild population), and that they carried much
of the standing genetic variance present in the population collected
from the field. We took six protonymphs (second juvenile stage) from
each from inbred line (hence, there were 72 founders per population) and
expanded them for two generations with random mating. The expanded
populations were then subdivided into four F populations and four S
populations by transferring 50 randomly selected protonymphs in a new
vial. Next generation, each of these F and S populations was further
subdivided it two treatments: gradual temperature increase (GTI, with
three replicates per subpopulation) and control (one replicate). The GTI
populations (FT and ST henceforth) were maintained at 25°C during first
generation, with temperature increasing for the next two generations by
2°C, and then maintained at 29°C from third generation on (we did not
increase the temperature further as our pilot study indicated that mites
are unable to adapt to temperature over 30°C when the temperature
increases 2°C per generation). Control populations (FC and SC
henceforth) were maintained at 23°C at all generations. Each generation
lasted 10 days, with c.a. three days needed for adult eclosion and 7
days for mating and reproduction (mating takes place immediately upon
emergence and eggs are laid continuously starting ca. 24 h after
mating). Each new generation was started by 50 randomly selected
protonymphs. At each generation, we recorded the number of females,
scrambler, and fighter males twice: immediately after adults eclosed and
at the end of the reproduction period. A population was considered to be
extinct if (i) the females did not lay any eggs, (ii) no larvae hatched
from the laid eggs, or (iii) no adults of one or both sexes survived to
adulthood.